Cyclical expression of the Notch/Wnt regulator Nrarp requires modulation by Dll3 in somitogenesis

Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rhpn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function.     

Ndrg4 is required for normal myocyte proliferation during early cardiac development in zebrafish

NDRG4 is a novel member of the NDRG family (N-myc downstream-regulated gene). The roles of NDRG4 in development have not previously been evaluated. We show that, during zebrafish embryonic development, ndrg4 is expressed exclusively in the embryonic heart, the central nervous system (CNS) and the sensory system. Ndrg4 knockdown in zebrafish embryos causes a marked reduction in proliferative myocytes and results in hypoplastic hearts. This growth defect is associated with cardiac phenotypes in morphogenesis and function, including abnormal heart looping, inefficient circulation and weak contractility. We reveal that ndrg4 is required for restricting the expression of versican and bmp4 to the developing atrioventricular canal. This constellation of ndrg4 cardiac defects phenocopies those seen in mutant hearts of heartstrings (hst), the tbx5 loss-of-function mutants in zebrafish. We further show that ndrg4 expression is significantly decreased in hearts with reduced tbx5 activities. Conversely, increased expression of tbx5 that is due to tbx20 knockdown leads to an increase in ndrg4 expression. Together, our studies reveal an essential role of ndrg4 in regulating proliferation and growth of cardiomyocytes, suggesting that ndrg4 may function downstream of tbx5 during heart development and growth.     

Nkx genes regulate heart tube extension and exert differential effects on ventricular and atrial cell number

Heart formation is a complex morphogenetic process, and perturbations in cardiac morphogenesis lead to congenital heart disease. NKX2-5 is a key causative gene associated with cardiac birth defects, presumably because of its essential roles during the early steps of cardiogenesis. Previous studies in model organisms implicate NKX2-5 homologs in numerous processes, including cardiac progenitor specification, progenitor proliferation, and chamber morphogenesis. By inhibiting function of the zebrafish NKX2-5 homologs, nkx2.5 and nkx2.7, we show that nkx genes are essential to establish the original dimensions of the linear heart tube. The nkx-deficient heart tube fails to elongate normally: its ventricular portion is atypically short and wide, and its atrial portion is disorganized and sprawling. This atrial phenotype is associated with a surplus of atrial cardiomyocytes, whereas ventricular cell number is normal at this stage. However, ventricular cell number is decreased in nkx-deficient embryos later in development, when cardiac chambers are emerging. Thus, we conclude that nkx genes regulate heart tube extension and exert differential effects on ventricular and atrial cell number. Our data suggest that morphogenetic errors could originate during early stages of heart tube assembly in patients with NKX2-5 mutations.     

Sex-specific repression of dachshund is required for Drosophila sex comb development

The origin of new morphological structures requires the establishment of new genetic regulatory circuits to control their development, from initial specification to terminal differentiation. The upstream regulatory genes are usually the first to be identified, while the mechanisms that translate novel regulatory information into phenotypic diversity often remain obscure. In particular, elaborate sex-specific structures that have evolved in many animal lineages are inevitably controlled by sex-determining genes, but the genetic basis of sexually dimorphic cell differentiation is rarely understood. In this report, we examine the role of dachshund (dac), a gene with a deeply conserved function in sensory organ and appendage development, in the sex comb, a recently evolved male-specific structure found in some Drosophila species. We show that dac acts during metamorphosis to restrict sex comb development to the appropriate leg region. Localized repression of dac by the sex determination pathway is necessary for male-specific morphogenesis of sex comb bristles. This pupal function of dac is separate from its earlier role in leg patterning, and Dac at this stage is not dependent on the pupal expression of Distalless (Dll), the main regulator of dac during the larval period. Dll acts in the epithelial cells surrounding the sex comb during pupal development to promote sex comb rotation, a complex cellular process driven by coordinated cell rearrangement. Our results show that genes with well-conserved developmental functions can be re-used at later stages in development to regulate more recently evolved traits. This mode of gene co-option may be an important driver of evolutionary innovations.     

Bef medaka mutant reveals the essential role of c-myb in both primitive and definitive hematopoiesis

Vertebrate hematopoiesis is characterized by two evolutionally conserved phases of development, i.e., primitive hematopoiesis, which is a transient phenomenon in the early embryo, and definitive hematopoiesis, which takes place in the later stages. Beni fuji (bef) was originally isolated as a medaka mutant that has an apparently reduced number of erythrocytes in its peripheral blood. Positional cloning revealed that the bef mutant has a nonsense mutation in the c-myb gene. Previous studies have shown that c-myb is essential for definitive hematopoiesis, and c-myb is now widely used as a marker gene for the onset of definitive hematopoiesis. To analyze the phenotypes of the bef mutant, we performed whole-mount in situ hybridization with gene markers of hematopoietic cells. The bef embryos showed decreased expression of α-globin and l-plastin, and a complete loss of mpo1 and rag1 expression, suggesting that the bef embryos had defects not only in erythrocytes but also in other myeloid cells, which indicates that their definitive hematopoiesis was aberrant. Interestingly, we observed a diminution in the number of primitive erythrocytes and a delay in the emergence of primitive macrophages in the bef embryos. These results suggest that c-myb also functions in the primitive hematopoiesis, potentially demonstrating a link between primitive and definitive hematopoiesis.     

Temporal expression and steroidal regulation of piRNA pathway genes (mael, piwi, vasa) during Silurana (Xenopus) tropicalis embryogenesis and early larval development

It has been extensively documented that exposure of amphibians and teleost fish to exogenous steroid hormones like estrogen, androgen, xenoestrogen or steroid biosynthesis inhibitors can impair their gonadal development or induce sex reversal against genotypic sex. However, the molecular pathways underlying sexual development and the effects of sex steroids or other exogenous hormones in these aquatic vertebrates remain elusive. Recently, a germ plasm-associated piRNA (piwi-interacting RNA) pathway has been shown to be a determinant in the development of animal gonadal germline cells. In the current study, we examined whether this piRNA pathway is involved in the regulation of sex steroid hormones in gonadal development. We firstly established developmental expression patterns of three key piRNA pathway genes (mael, piwi and vasa), during Silurana (Xenopus) tropicalis embryogenesis and early larval development. All three genes exhibit high expression at early developmental stages and have significantly decreased expression thereafter, indicating a very active involvement of piRNA pathway at the beginning of embryogenesis. We further examined gene expression changes of those genes in frog larvae exposed to two sex steroid biosynthesis inhibitors, fadrozole and finasteride, both of which are known to result in male-biased or female-biased phenotypes, respectively. We found that fadrozole and finasteride exposures increased the expression of piRNA pathway genes such as mael and vasa at the larval stage when the expression of piRNA pathway genes is programmed to be very low. Therefore, our results indicate that the piRNA pathway is likely a common pathway by which different sex steroid hormones regulate gonadal sex differentiation.     

Retinoic acid signaling in perioptic mesenchyme represses Wnt signaling via induction of Pitx2 and Dkk2

Morphogenesis during eye development requires retinoic acid (RA) receptors plus RA-synthesizing enzymes, and loss of RA signaling leads to ocular disorders associated with loss of Pitx2 expression in perioptic mesenchyme. Several Wnt signaling components are expressed in ocular tissues during eye development including Dkk2, encoding an inhibitor of Wnt/β-catenin signaling, which was previously shown to be induced by Pitx2 in the perioptic mesenchyme. Here, we investigated potential cross-talk between RA and Wnt signaling during ocular development. Genetic studies using Raldh1/Raldh3 double null mice deficient for ocular RA synthesis demonstrated that Pitx2 and Dkk2 were both down-regulated in perioptic mesenchyme. Chromatin immunoprecipitation and gel mobility shift studies demonstrated the existence of a DR5 RA response element upstream of Pitx2 that binds all three RA receptors in embryonic eye. Axin2, an endogenous readout of Wnt/β-catenin signaling, was up-regulated in cornea and perioptic mesenchyme of RA deficient embryos. Also, expression of Wnt5a was expanded in perioptic mesenchyme of RA deficient eyes. Our findings demonstrate excessive activation of Wnt signaling in the perioptic mesenchyme of RA deficient mice which may be responsible for abnormal development leading to defective optic cup, cornea, and eyelid morphogenesis.     

Bestrophin genes are expressed in Xenopus development

The Bestrophin-1/VMD2 gene has been implicated in Best disease, a juvenile-onset vitelliform macular dystrophy. The Bestrophin proteins have anion channel activity, and the four mammalian members share sequence homologies in multiple transmembrane domains and an RFP-tripeptide motif. The expression patterns and functions of the Bestrophin genes in retinal pigment epithelium have been studied intensively, whereas little is known about their roles in vertebrate embryogenesis. This study examined the roles of four Xenopus tropicalis homologs of BEST genes. The xtBest genes showed spatially and temporally distinct expression. xtBest-2 was the only maternally expressed Best gene, and both xtBest-2 and the Xenopus laevis Best-2 gene were expressed at the edge of the blastopore lip including the organizer. Ectopic expression of xBest-2 caused defects in dorsal axis formation and in mesodermal gene expression during gastrulation. These results suggest a new role of the Bestrophin family genes in early vertebrate embryogenesis.     

Conditional Aurora A deficiency differentially affects early mouse embryo patterning

Aurora A is a mitotic kinase essential for cell proliferation. In mice, ablation of Aurora A results in mitotic arrest and pre-implantation lethality, preventing studies at later stages of development. Here we report the effects of Aurora A ablation on embryo patterning at early post-implantation stages. Inactivation of Aurora A in the epiblast or visceral endoderm layers of the conceptus leads to apoptosis and inhibition of embryo growth, causing lethality and resorption at approximately E9.5. The effects on embryo patterning, however, depend on the tissue affected by the mutation. Embryos with an epiblast ablation of Aurora A properly establish the anteroposterior axis but fail to progress through gastrulation. In contrast, mutation of Aurora A in the visceral endoderm, leads to posteriorization of the conceptus or failure to elongate the anteroposterior axis. Injection of ES cells into Aurora A epiblast knockout blastocysts reconstitutes embryonic development to E9.5, indicating that the extra-embryonic tissues in these mutant embryos can sustain development to organogenesis stages. Our results reveal new ways to induce apoptosis and to ablate cells in a tissue-specific manner in vivo. Moreover, they show that epiblast-ablated embryos can be used to test the potency of stem cells.     

The G314S KCNQ1 mutation exerts a dominant-negative effect on expression of KCNQ1 channels in oocytes

Congenital long QT syndrome is a cardiac disorder characterized by prolongation of QT interval on the surface ECG associated with syncopal attacks and a high risk of sudden death. Mutations in the voltage-gated potassium channel subunit KCNQ1 induce the most common form of long QT syndrome (LQT1). We previously identified a hot spot mutation G314S located within the pore region of the KCNQ1 ion channel in a Chinese family with long QT syndrome. In the present study, we used oocyte expression of the KCNQ1 polypeptide to study the effects of the G314S mutation on channel properties. The results of electrophysiological studies indicate G314S, co-expressed with KCNE1 was unable to assemble to form active channel. G314S, co-expressed with WT KCNQ1 and KCNE1, suppressed I_ks currents in a dominant-negative manner, which is consistent with long QT syndrome in the members of the Chinese family carrying G314S KCNQ1 mutation.     

The identification of microRNAs in the whitespotted bamboo shark (Chiloscyllium plagiosum) liver by Illumina sequencing

MicroRNAs are indispensable players in the regulation of a broad range of biological processes. Here, we report the first deep sequencing of the whitespotted bamboo shark (Chiloscyllium plagiosum) liver. We mapped 91 miRNAs in the Callorhinchus milii genome that have previously been described in the Danio rerio, Fugu rubripes, Oryzias latipes, Xenopus laevis, Xenopus tropicalis, Homo sapiens, and Mus musculus. In addition, 156 new putative candidate (PC) C. plagiosum miRNAs were identified. From these 247 miRNAs, 39 miRNA clusters were identified, and the expression of these clustered miRNAs was observed to vary significantly. A total of 7 candidate miRNAs were selected for expression confirmation by stem-loop RT-PCR. This study resulted in the addition of a significant number of novel miRNA sequences to GenBank and laid the foundation for further understanding of the function of miRNAs in the regulation of C. plagiosum liver development.